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Dual genetic tracing reveals a unique fibroblast subpopulation modulating cardiac fibrosis

Nature Genetics volume 55, pages 665–678 (2023)Cite this article

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Abstract

After severe heart injury, fibroblasts are activated and proliferate excessively to form scarring, leading to decreased cardiac function and eventually heart failure. It is unknown, however, whether cardiac fibroblasts are heterogeneous with respect to their degree of activation, proliferation and function during cardiac fibrosis. Here, using dual recombinase-mediated genetic lineage tracing, we find that endocardium-derived fibroblasts preferentially proliferate and expand in response to pressure overload. Fibroblast-specific proliferation tracing revealed highly regional expansion of activated fibroblasts after injury, whose pattern mirrors that of endocardium-derived fibroblast distribution in the heart. Specific ablation of endocardium-derived fibroblasts alleviates cardiac fibrosis and reduces the decline of heart function after pressure overload injury. Mechanistically, Wnt signaling promotes activation and expansion of endocardium-derived fibroblasts during cardiac remodeling. Our study identifies endocardium-derived fibroblasts as a key fibroblast subpopulation accounting for severe cardiac fibrosis after pressure overload injury and as a potential therapeutic target against cardiac fibrosis.

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Fig. 1: Distribution of endocardium-derived fibroblasts in the adult heart.
Fig. 2: Endocardium-derived fibroblasts expand regionally during cardiac fibrosis after TAC.
Fig. 3: Contribution of epicardium-derived fibroblasts to cardiac fibrosis after TAC.
Fig. 4: Fibroblast-specific proliferation tracing reveals regional fibroblast proliferation after TAC.
Fig. 5: Genetic ablation of endocardium-derived fibroblasts alleviates cardiac fibrosis and reduces the decline of heart function after TAC.
Fig. 6: Gene profiles of endocardium- and epicardium-derived fibroblasts in adult hearts after TAC.
Fig. 7: Ctnnb1 knockout reduces cardiac fibrosis and the decline of heart function after TAC.
Fig. 8: Endocardium-derived fibroblasts expand after TAC and targeting them alleviates cardiac fibrosis.

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Data availability

The raw sequencing data reported in this study can be accessed at the NCBI Sequence Read Archive under BioProject no. PRJNA813322.

Code availability

This study did not generate any unique code or algorithm. The algorithms used for the analysis in this study are all publicly available. The code used for single-cell data processing and analysis has been deposited in Zenodo (https://doi.org/10.5281/zenodo.7593543).

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Acknowledgements

This study was supported by the National Key Research & Development Program of China (grant nos. 2019YFA0110403 and 2019YFA0802000 to B.Z., grant no. 2022YFA1104200 to Y.L. and grant nos. 2021YFA0805100 and 2018YFA0108100 to L.H.), National Science Foundation of China (grant nos. 82088101 and 32050087 to B.Z., grant nos. 82270415 and 81970412 to L.W. and grant no. 32170848 to Y.L.), Shanghai Pilot Program for Basic Research-CAS, Shanghai Branch (grant no. JCYJ-SHFY-2021-0 to B.Z.), CAS Project for Young Scientists in Basic Research (grant no. YSBR-012 to B.Z.), the Pearl River Talent Recruitment Program of Guangdong Province (grant no. 2017ZT07S347 to B.Z.), Young Elite Scientists Sponsorship Program Grant of CAS (to W.P. and Y.L.), Research Grants Council of Hong Kong (grant no. RFS2223-4S04 to K.O.L.), Research Funds of Hangzhou Institute for Advanced Study (grant no. 2022ZZ01015 to X. Li), Collaborative Innovation Program of Shanghai Municipal Health Commission (grant no. 2020CXJQ01 to B.Z.), Program of Shanghai Academic Research Leader (grant no. 22XD1403700 to B.Z.), the XPLORER PRIZE (to B.Z.), the New Cornerstone Science Foundation (to B.Z.), Shanghai Youth Science and Technology Rising-Star Program (to Y.L.), AstraZeneca, Boehringer Ingelheim and Sanofi-Shanghai Institutes for Biological Sciences Fellowship. We also thank Shanghai Model Organisms Center and Cyagen Biosciences for generating the mice; members of the animal facility and cell platform in Center for Excellence in Molecular Cell Science (CEMCS) and National Center for Protein Science Shanghai for assistance in microscopy; and the Genome Tagging Project Center, CEMCS, CAS for technical support.

Author information

Author notes

  1. These authors contributed equally: Maoying Han, Zixin Liu, Lei Liu.

Authors and Affiliations

  1. State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences University of the Chinese Academy of Sciences, Shanghai, China

    Maoying Han, Zixin Liu, Xiuzhen Huang, Haixiao Wang, Wenjuan Pu, Xiuxiu Liu, Yan Li & Bin Zhou

  2. School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    Maoying Han, Lei Liu & Bin Zhou

  3. Department of Vascular Surgery, Zhongshan Hospital, Fudan University, Shanghai, China

    Enci Wang & Lixin Wang

  4. School of Life Sciences, Westlake University, Hangzhou, China

    Lingjuan He

  5. Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of the Chinese Academy of Sciences, Hangzhou, China

    Xufeng Li & Bin Zhou

  6. Chinese Aacademy of Sciences Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of Nutrition and Health, University of the Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing, China

    Jiayu Wu & Lin Qiu

  7. Shanghai Laboratory Animal Research Center, Shanghai, China

    Ruling Shen

  8. Bioscience Cardiovascular, Research and Early Development, Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden

    Qing-Dong Wang

  9. The Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, China

    Yong Ji & Bin Zhou

  10. Division of Cardiology, Department of Medicine, Baylor College of Medicine, Houston, TX, USA

    Reza Ardehali

  11. Department of Pediatric Cardiaovascular Surgery, Children’s Hospital, School of Medicine, Zhejiang University, National Clinical Research Center for Child Health, Hangzhou, China

    Qiang Shu

  12. Department of Chemical Pathology and Li Ka Shing Institute of Health Sciences, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China

    Kathy O. Lui

  13. Department of Vascular Surgery, Zhongshan Hospital (Xiamen), Fudan University, Xiamen, China

    Lixin Wang

  14. New Cornerstone Science Laboratory, Shenzhen, China

    Bin Zhou

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  1. Maoying Han
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Contributions

M.H. and B.Z. conceived and designed the project. Z.L. performed the RNA sequencing experiments and analysis. L.L., X.H., H.W., W.P., E.W., X. Liu, Y.L., L.H., X. Li, J.W., L.Q., R.S., Q.-D.W., Y.J., R.A., Q.S. and L.W. bred the mice, performed the experiments, analyzed the data or made intellectual contributions to the study. K.O.L. and B.Z. drafted and revised the paper.

Corresponding author

Correspondence to Bin Zhou.

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Nature Genetics thanks Xing Fu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Generation and characterization of R26-RL-GFP mouse line.

a, Schematic figure showing the knock-in strategy for R26-RL-GFP by homologous recombination using CRISPR/Cas9. b, Whole-mount bright-field and epifluorescence images of multiple organs collected from adult R26-RL-GFP mouse. c, Schematic figure showing the knock-in strategy of Tnnt2-Dre-Cre-tdTomato (Tnnt2-DCT) by homologous recombination. d, Schematic diagram showing the Cre-loxP and Dre-rox recombinations after crossing R26-RL-GFP with Tnnt2-DCT mice. e, Whole-mount bright-field and epifluorescence images of Tnnt2-DCT;R26-RL-GFP mouse heart (left panel). Immunostaining for TNNI3 and GFP on heart sections (right panel). f, Quantification of the percentage of TNNI3+ cardiomyocytes expressing GFP on Tnnt2-DCT;R26-RL-GFP heart sections. Data are mean ± s.e.m.; n = 5 mice; *P < 0.0001. Statistical analysis was performed by unpaired Student’s t-test. g, Schematic diagram showing the Dre-rox or Cre-loxP recombinations after crossing R26-RL-GFP with CAG-Dre or ACTB-Cre. h, Whole mount bright-field and epifluorescence images of CAG-Dre;R26-RL-GFP and ACTB-Cre;R26-RL-GFP mouse heart (left panel). Immunostaining for GFP on heart sections (right panel). i, Quantification of the percentage of GFP+ cells on mouse heart sections. Data are mean ± s.e.m.; n = 5 mice. V, ventricle; D, dorsal. Scale bars, yellow, 1 mm; white, 100 µm. Each image is representative of five individual mouse samples.

Extended Data Fig. 2 Labeling of cardiac cells by Nfatc1-Dre.

a, Schematic figure showing the experimental strategy. b, Immunostaining for WT1 and tdTomato on embryo sections collected from Nfatc1-Dre;R26-RSR-tdTomato mice. The boxed areas are magnified on the right panel with split channels. Arrowheads: white, tdTomato+ cells; yellow, WT1+ epicardium cells. c, Quantification of the percentage of tdTomato+ cells expressing WT1. Data are mean ± s.e.m.; n = 5 mice. d, Schematic figure showing the experimental strategy. e,f, Immunostaining for CD31, PDGFRa, and tdTomato on embryonic sections collected from Nfatc1-Dre;R26-RSR-tdTomato mice. g, Quantification of the percentage of tdTomato+ cells expressing PDGFRa or CD31 on the outer layer of hearts collected from Nfatc1-Dre;R26-RSR-tdTomato mice. Data are mean ± s.e.m.; n = 5 mice h, Immunostaining for PDGFRa and GFP on adult heart sections collected from Nfatc1-Dre;Col1a2-CreER;R26-RL-GFP mice. The boxed areas are magnified on the lower panels. i, Quantification of the percentage of PDGFRa+ cells expressing GFP of the outer layer of hearts. Data are mean ± s.e.m.; n = 5 mice. Scale bars, yellow, 1 mm, white, 100 µm. Each image is representative of five individual mouse samples.

Extended Data Fig. 3 Genetic labeling of endocardium-derived fibroblasts (EndoFb) in developing heart.

a, Illustration of the intersectional genetic approach for genetic labeling of EndoFb. b, Schematic figure showing the strategy for labelling the EndoFb by tamoxifen (Tam) treatment. c, Cartoon image showing different region of embryonic heart. RV, right ventricle; VS, ventricular septum; LV, left ventricle. d, Whole-mount (inset) and epifluorescence images of hearts at different embryonic stages show the expression pattern of EndoFb. e,g,i, Immunostaining for PDGFRa and GFP on heart sections of different embryonic stages. The boxed areas are magnified on the right panels with split channels. Arrowheads, GFP+PDGFRa+ EndoFb. f,h,j, Cartoon images showed the distribution of EndoFb in hearts at different embryonic stages. k, Quantification of the percentage of PDGFRa+ fibroblasts expressing GFP in E11.5 hearts. Data are mean ± s.e.m.; n = 5 mice. l,m, Quantification of the percentage for PDGFRa+ EndoFb expressing GFP in E14.5 and E17.5 hearts. Data are mean ± s.e.m.; n = 5 mice. RCM, right compact myocardium; RTM, right trabecular myocardium; LCM, left compact myocardium; LTM, left trabecular myocardium; VS, ventricular septum. n, Cartoon image showed EndoFb from EndoMT and their subsequent migration into some regions of embryonic hearts. Scale bars, 100 µm. Each image is representative of five individual mouse samples.

Extended Data Fig. 4 Temporospatial determination of EndoFb fate in developing heart.

a, Schematic diagram showing the strategy for labelling the EndoFb in different time window. 4-Hydroxytamoxifen (4-OH Tam) was injected at E7.5, E11.5, E12.5, and E16.5, then collected at E17.5. b, Whole-mount and epifluorescence images of E17.5 hearts with Tam induced at different time points. c-f, Immunostaining for PDGFRa and GFP on E17.5 heart sections when 4-OH Tam was injected at E7.5, E11.5, E12.5, and E16.5 respectively. Arrowheads, GFP+PDGFRa+ EndoFb. g, Quantification of the percentage of GFP+ EndoFb expressing PDGFRa in total (left panel), different valves (middle panel), and different regions of ventricles (right panel) in different time window. Data are mean ± s.e.m., n = 5 mice. Two-way ANOVA followed by Tukey’s multiple comparisons test was used to compare among means of multiple groups. In All, E7.5 > E17.5 vs. E11.5 > E17.5 and E11.5 > E17.5 vs. E12.5 > E17.5, *P < 0.0001. In Valve, PL: E7.5 > E17.5 vs. E11.5 > E17.5 and E11.5 > E17.5 vs. E12.5 > E17.5, *P < 0.0001; SL: E7.5 > E17.5 vs. E11.5 > E17.5 and E11.5 > E17.5 vs. E12.5 > E17.5, *P < 0.0001; AL: E7.5 > E17.5 vs. E11.5 > E17.5 and E11.5 > E17.5 vs. E12.5 > E17.5, *P < 0.0001; ML: E7.5 > E17.5 vs. E11.5 > E17.5, *P < 0.0001; E11.5 > E17.5 vs. E12.5 > E17.5, *P = 0.0002. In Myocardium, RCM: E7.5 > E17.5 vs. E11.5 > E17.5, *P = 0.0226; RTM: E7.5 > E17.5 vs. E11.5 > E17.5, *P < 0.0001; LCM: E7.5 > E17.5 vs. E11.5 > E17.5, *P < 0.0001; LTM: E7.5 > E17.5 vs. E11.5 > E17.5, *P < 0.0001; VS: E7.5 > E17.5 vs. E11.5 > E17.5 and E11.5 > E17.5 vs. E12.5 > E17.5, *P < 0.0001; ns, no significance. PL, parietal leaflet; SL, septal leaflet; AL, aortic valve; ML, mural valve; RCM, right compact myocardium; RTM, right trabecular myocardium; LCM, left compact myocardium; LTM, left trabecular myocardium; VS, ventricular septum. Scale bars: yellow, 200 µm; white, 100 µm. Each image is representative of five individual mouse samples.

Extended Data Fig. 5 Expression of fibroblast markers in GFP+ EndoFb.

a, Schematic diagram showing the strategy for labelling the EndoFb. b, Immunostaining for PDGFRa and GFP on Nfatc1-Dre;Col1a2-CreER;R26-RL-GFP heart sections. The boxed area was magnified on the right panel. c, Quantification of the percentage of GFP+ cells expressing PDGFRa (left panel), and the percentage of PDGFRa+ cells expressing GFP (right panel). d, Z-stack confocal images of heart sections stained for Vimentin and GFP. YZ and XZ indicated signals from dotted lines on Z-stack images. e, Quantification of the percentage of GFP+ cells expressing Vimentin (left panel) and the percentage of Vimentin+ cells expressing GFP (right panel). f, Immunostaining for Vimentin and PDGFRa on heart sections. g, Quantification of the percentage of PDGFRa+ cells expressing Vimentin. h, Immunostaining for aSMA and GFP on heart sections. The boxed areas were magnified on the right panel. YZ and XZ indicated signals from dotted lines on Z-stack images. i, Quantification of the percentage of GFP+ cells expressing aSMA. Scale bars, 100 µm. All quantification data are represented as mean ± s.e.m.; n = 5 mice. Each image is representative of five individual mouse samples.

Extended Data Fig. 6 Distribution of EndoFb in hearts at 28 days post TAC.

a, Schematic diagram showing the experimental strategy. b, Whole-mount bright-field and fluorescent views of hearts collected from Nfatc1-Dre;Col1a2-CreER;R26-RL-GFP mice at 28d post sham or TAC. c, Sirius Red staining images showing the fibrotic area on heart sections collected from mice post TAC 28 days. d, Immunostaining for PDGFRa, Vimentin, aSMA, and GFP on whole heart sections. e-g, Immunostaining for PDGFRa (e), Vimentin (f), aSMA (g), with EdU and GFP on VS and LV of the heart sections. Scale bars: yellow, 1 mm; white, 100 µm. Each image is representative of five individual mouse samples.

Extended Data Fig. 7 Efficient genetic ablation of EndoFb.

a, Schematic diagram showing the experimental strategy. b, Whole-mount bright-field and fluorescent views of hearts collected from mice after PBS- or DT-treatment. c, Immunostaining for tdTomato on whole heart sections collected from mice with PBS- or DT-treatment. d, Immunostaining for tdTomato and PDGFRa on the different parts of the heart sections collected from mice with PBS- or DT-treatment. e, Quantification of the PDGFRa+ fibroblasts expressing tdTomato in different areas of hearts collected from mice treated with PBS or DT. Data are mean ± s.e.m.; n = 5 mice; in every area, PBS vs. DT, *P < 0.0001. f, Immuostaining for DTR and tdTomato on hearts collected from mice treated with PBS or DT. g, Quantification of the percentage of the tdTomato+ cells of hearts collected from mice treated with PBS or DT. Data are mean ± s.e.m.; n = 5 mice; *P < 0.0001. h,i, Quantification of ejection fraction (EF) and fractional shortening (FS) of mice treated with PBS or DT. Data are mean ± s.e.m.; n = 5 mice; ns, no significance. Scale bars: yellow, 1 mm; white, 100 µm. Each image is representative of five individual mouse samples. Two-way ANOVA followed by Sidak’s multiple-comparisons test was used in e. Unpaired Student’s t-test was performed in g-i.

Extended Data Fig. 8 Gene profile of EndoFb and EpiFb after injury.

a, Schematic diagram showing two experimental designs. b, Gene expression heatmap showing the scaled expression of selected GO pathway-related genes in GFP+ and GFP– fibroblasts from Sham or TAC hearts (Experiment 1). c, Venn plots showing the number of shared significantly up-regulated genes between the comparison groups from Experiment 1 and 2. d, GO pathway enrichment analysis. Each row represents a GO term, and each column represents a comparison group. White cell denotes that gene changes are not enriched in this GO pathway between the comparison groups. Over-representation test was used in d; n = 3 mice.

Extended Data Fig. 9 Characterization of Col1a2-RSR-CreER mouse.

a, Schematic figure showing rox-flanked Stop cassette removal from Col1a2-RSR-CreER and R26-RL-GFP by Nfatc1-Dre, and loxP-flanked Stop cassette removal from R26-RL-GFP by Col1a2-RSR-CreER after injecting tamoxifen (Tam). b, Schematic figure showing the experimental design. c, Immunostaining of PDGFRa and GFP on whole heart sections. d, Quantification of the percentage of GFP+ cells expressing PDGFRa in different regions of hearts. Data are mean ± s.e.m.; n = 5 mice. e-h, Magnifications of different regions on heart sections. VS, ventricular septum; RV, right ventricle; LV, left ventricle; OMW: outer myocardium wall; IMW, inner myocardium wall. Scale bar: yellow, 1 mm; white, 100 µm. Each image is representative of five individual mouse samples.

Extended Data Fig. 10 Knockout of Ctnnb1 in EndoFb by dual recombinases.

a, Schematic figure showing the experimental design. b, qRT-PCR of Ctnnb1 expression in GFP– and GFP+ fibroblasts sorted from the mutant mice. Data are mean ± s.e.m.; n = 5 mice; *P < 0.0001. c, d, Quantification of ejection fraction (EF) and fractional shortening (FS) of the control and mutant mice. Data are mean ± s.e.m.; n = 5 mice; ns, no significance. e, Immunostaining for PDGFRa, β-catenin, and GFP on different parts of hearts collected from the mutant mice. Yellow arrowheads, GFP– β-catenin+ PDGFRa+ cells; white arrowheads, GFP+ β-catenin– PDGFRa+ cells. Scale bars, 100 µm. Each image is representative of five individual mouse samples. Unpaired Student’s t-test was performed in b-d.

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Han, M., Liu, Z., Liu, L. et al. Dual genetic tracing reveals a unique fibroblast subpopulation modulating cardiac fibrosis. Nat Genet 55, 665–678 (2023). https://doi.org/10.1038/s41588-023-01337-7

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